The long term goal of this research is to obtain a better understanding of the origins of enzyme specificity and catalysis and how loop regions and residues distal to the active site contribute to catalysis and substrate selectivity. Our research utilizes two enzymes that are members of the same superfamily, cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and a cis-CaaD homologue designated CgX. These enzymes share 54% sequence similarity and the key elements of the catalytic machinery. Yet, CgX is a poor cis-CaaD as reflected by its low efficiency (processing the cis-isomer) and lack of specificity (processing both isomers). The reasons for these differences are not known, yet this information could shed light on the evolution of isomer specificity in the cis- and frans-3-chloroacrylic acid dehalogenases, lead to a better understanding of the characteristics of the tautomerase superfamily, and advance our knowledge of enzymes. The aims of this proposal will assess the influence of protein motion, loop identity, and distal hydrogen bonding networks involving active site residues on each step of the catalytic cycle. Our two major aims are (1) to determine the effects of the individual replacement of the six amino acids of the flexible cis-CaaD active site loop with those of CgX on both steady-state and pre-steady state kinetic parameters;and (2) to examine the role of the critical cis-CaaD residue His28 in substrate binding by investigating the effects of indirect and direct mutation(s) on the kinetics of both binding and catalysis. Kinetic data will be globally fit to a single mechanism by simulation to resolve individual rate constants. This approach will allow the effects of mutational replacements on binding, chemistry, conformational changes, and/or product release steps to be critically evaluated. These studies, combined with other results, could form the basis for a well-defined model for the study of enzyme specificity and become a valuable resource for developing new approaches to combat antibiotic resistance. PUBLIC HEALTH RELEVANCE: Antibiotic resistant bacteria have become a major public health threat and will continue to be one. It is therefore critical to understand the origins of enzyme specificity and how it changes as a result of mutations;as this is one way bacteria acquire abilities that render them less susceptible to antibiotic treatments.